The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by γ-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to γ-irradiation-induced cell death. LNCaP cells can be sensitized to γ-irradiation by tumor necrosis factor α (TNF-α) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-α activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-α, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysine-chloromethylketone), but not by the caspase inhibitor z-VAD (2–val–Ala–Asp (oMe)–CH 2 F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8,-3, or-7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-α, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-α with or without γ-irradiation. TNF-α and γ-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.